Timing and localization of myasthenia gravis-related gene expression.
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ABSTRACT: Myasthenia gravis (MG) is an acquired autoimmune disorder caused by autoantibodies binding acetylcholine receptors (AChR), muscle-specific kinase (MuSK), agrin or low-density lipoprotein receptor-related protein 4 (Lrp4). These autoantibodies inhibit neuromuscular transmission by blocking the function of these proteins and thereby cause fluctuating skeletal muscle weakness. Several reports suggest that these autoantibodies might also affect the central nervous system (CNS) in MG patients. A comprehensive overview of the timing and localization of the expression of MG-related antigens in other organs is currently lacking. To investigate the spatio-temporal expression of MG-related genes outside skeletal muscle, we used in silico tools to assess public expression databases. Acetylcholine esterase, nicotinic AChR α1 subunit, agrin, collagen Q, downstream of kinase-7, Lrp4, MuSK and rapsyn were included as MG-related genes because of their well-known involvement in either congenital or autoimmune MG. We investigated expression of MG-related genes in (1) all human tissues using GTEx data, (2) specific brain regions, (3) neurodevelopmental stages, and (4) cell types using datasets from the Allen Institute for Brain Sciences. MG-related genes show heterogenous spatio-temporal expression patterns in the human body as well as in the CNS. For each of these genes, several (new) tissues, brain areas and cortical cell types with (relatively) high expression were identified suggesting a potential role for these genes outside skeletal muscle. The possible presence of MG-related antigens outside skeletal muscle suggests that autoimmune MG, congenital MG or treatments targeting the same proteins may affect MG-related protein function in other organs.
Project description:Background Myasthenia gravis (MG) is an autoimmune disorder with fluctuating muscle weakness, divided into generalized and localized (ocular) forms. Maternal antibodies to acetylcholine receptors cross the placenta and may cause transient neonatal myasthenia gravis (TNMG). We present a case of seronegative maternal ocular MG and delayed TNMG. Case A 29-year-old G3P1011 underwent cesarean birth of a male infant who developed oxygen desaturation requiring supplemental oxygen on day of life (DOL) 3. Based on the clinical course and after exclusion of other diagnoses, the infant was diagnosed with TNMG. Infant's condition improved spontaneously and he was weaned off supplemental oxygen and discharged home on DOL 12. Conclusion Infants born to mothers with seronegative localized (ocular) MG are also susceptible to TNMG which may be late in onset.
Project description:ObjectiveWe sought to identify a causative mutation in a previously reported kindred with parental consanguinity and 5 of 10 siblings with adult-onset autoimmune myasthenia gravis.MethodsWe performed genome-wide homozygosity mapping, and sequenced all known genes in the one region of extended homozygosity. Quantitative and allele-specific reverse transcriptase PCR (RT-PCR) were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles in a heterozygote.ResultsA region of shared homozygosity at chromosome 13q13.3-13q14.11 was found in 4 affected siblings and 1 unaffected sibling. A homozygous single nucleotide variant was found in the 3'-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that expression of ENOX1 decreased to about 20% of normal levels in lymphoblastoid cells from individuals homozygous for the variant and to about 50% in 2 unaffected heterozygotes. Allele-specific RT-PCR showed a 55%-60% reduction in the level of the variant transcript in heterozygous cells due to reduced mRNA stability.ConclusionThese results indicate that this sequence variant in ENOX1 may contribute to the familial autoimmune myasthenia in these patients.
Project description:BackgroundUnbiased in silico approaches applied to genome-wide data prioritized putative functional gene variants associating with treatment-resistant ophthalmoplegic myasthenia gravis (OP-MG). Although altered expression of genes harbouring these variants, or associated pathways, were shown in patient-derived transdifferentiated-myocyte models, gene expression in orbital-derived muscle was required to test the validity of the predictions.MethodsWe sampled orbicularis oculi muscle (OOM) and one paralysed extraocular muscle (EOM) from six individuals with OP-MG during blepharoptosis and re-alignment surgeries, respectively. For controls, the OOMs were sampled from four individuals without myasthenia undergoing surgery for non-muscle causes of ptosis, and one non-paralysed EOM. Using a qPCR array, expression of 120 genes was compared between OP-MG and control OOMs, profiling putative "OP-MG" genes, genes in related biological pathways and genes reported to be dysregulated in MG cases or experimental MG models, and in EOMs of cases with strabismus. Normalization was performed with two stable reference genes. Differential gene expression was compared between OP-MG and control samples using the ΔΔCT method. Co-expression was analysed by pairwise correlation of gene transcripts to infer expression networks.ResultsOverall, transcript levels were similar in OOMs and EOMs (p = 0.72). In OOMs, significant downregulated expression of eight genes was observed in OP-MG cases compared with controls (> twofold; p ≤ 0.016), including TFAM, a mitochondrial transcription factor, and genes related to the following pathways: atrophy signalling; muscle regeneration and contraction; glycogen synthesis; and extracellular matrix remodelling. Several microRNAs, known to be highly expressed in EOMs, are predicted to regulate some of these genes. Co-expression analyses of gene-pairs suggested high interconnectedness of gene expression networks in OP-MG muscle, but not controls (r > 0.96, p < 0.01). Significant inverse directions of gene-pair correlations were noted in OP-MG versus controls OOM networks (r ≥ 0.92, p < 0.001) involving most OP-MG genes overlapping prominently with muscle atrophy/contractility and oxidative metabolism genes.ConclusionsThe gene expression in orbital muscles derived from OP-MG individuals compared with normal controls, support the pathogenic hypothesis previously generated from whole genome sequence analyses. Repression of gene transcripts in OP-MG orbital muscle implicate tissue-specific regulatory mechanisms, which may inform future biomarker discovery approaches.
Project description:BackgroundThe pathogenesis of extraocular muscle (EOM) weakness in myasthenia gravis might involve a mechanism specific to the EOM. The aim of this study was to investigate characteristics of the EOM related to its susceptibility to myasthenia gravis.MethodsFemale F344 rats and female Sprague-Dawley rats were assigned to experimental and control groups. The experimental group received injection with Ringer solution containing monoclonal antibody against the acetylcholine receptor (AChR), mAb35 (0.25 mg/kg), to induce experimental autoimmune myasthenia gravis, and the control group received injection with Ringer solution alone. Three muscles were analyzed: EOM, diaphragm, and tibialis anterior. Tissues were examined by light microscopy, fluorescence histochemistry, and transmission electron microscopy. Western blot analysis was used to assess marker expression and ELISA analysis was used to quantify creatine kinase levels. Microarray assay was conducted to detect differentially expressed genes.ResultsIn the experimental group, the EOM showed a simpler neuromuscular junction (NMJ) structure compared to the other muscles; the NMJ had fewer synaptic folds, showed a lesser amount of AChR, and the endplate was wider compared to the other muscles. Results of microarray assay showed differential expression of 54 genes in the EOM between the experimental and control groups.ConclusionVarious EOM characteristics appear to be related to the increased susceptibility of the EOM and the mechanism of EOM weakness in myasthenia gravis.
Project description:Autoimmune myasthenia gravis (MG) is characterized by thymic abnormalities such as hyperplasia and thymoma. Thymus plays an important role in self-tolerance and is involved in initiation and progression of the disease. A large proportion of MG patient show the presence of ectopic germinal centers (GC) in thymus that are absent in normal individuals. However, the exact mechanism how this change in thymus morphology is triggered and if this is related to pathophysiology of the disease remains unknown. In this study we have compared the mRNA profile of thymus samples obtained during a clinical trial (Thymectomy Trial in Non-Thymomatous Myasthenia Gravis Patients Receiving Prednisone Therapy. ClinicalTrials.gov Identifier: NCT00294658). We have compared the mRNA profile of thymus samples that have distinct germinal centers with those that lack them using Affymetrix human transcriptome array 2.0 to identify the differentially expressed genes with in the two groups.
Project description:Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction, whose clinical hallmark is muscle weakness and early fatigability. Azathioprine (AZA) is commonly used in Myasthenia Gravis therapy. AZA is a purine antagonist, which inhibits the cell cycle in the resting and DNA synthesis phases. It is usually used as an immunosuppressant to block T- and B-cell proliferation. AZA, bioconverted to 6-mercaptopurine by glutathione S-transferase (GST), can be metabolized either through the hypoxanthine phosphoribosyl transferase pathway to active 6-thioguanine nucleotides (6-TGN) or through the thiopurine S-methyltransferase (TPMT) pathway to inactive methyl-thiopurine metabolites. The incorporation of active 6-TGNs into DNA, causing breaks in DNA strands resulting in interference with RNA production and thereby protein synthesis, is responsible for the drug effect. The response to AZA is determined by which metabolic pathway is being favored. While balanced use of both HPRT and TPMT pathways results in responsiveness to AZA, hyperactivity of TPMT skews the balance towards the TPMT pathway and results in unresponsiveness to AZA with no pharmacological effect. In contrast, low TPMT activity skews the balance towards the HPRT pathway, resulting in increasing side-effects due to the accumulation of 6-TGNs. Current treatments for MG therapy are often inadequate because less than 40% of patients achieve complete remission with available drugs. This reflects the lack of drugs acting on target sites for which there is strong evidence of pathogenicity and the inability to identify responder patients. No criteria for responsiveness are available, exposing patients to unpredictable failures and unpredictable side effects. These individuals are at particular risk for adverse drug reaction (ADRs) or therapeutic failure. Genetic profiling with the Affymetrix drug metabolizing enzymes and transports genotyping array offers the ability to determine 1,931 variants and 225 genes involved in drug metabolism and disposition. Accordingly, the study of well-known drug-metabolizing genes can be involved in the specific metabolic pathway of a drug, which is more likely to define genotype-phenotype association and thereby genotype profiles relevant to drug response that can be applied as predictive biomarkers for pharmacological treatment.
Project description:Background: Approximately 50% of thymoma patients also show myasthenia gravis (MG), which is an autoimmune disease; however, the pathogenesis of MG-associated thymoma remains elusive. Our aim was to investigate immune-related lncRNA profiles of a set of candidate genes for better understanding of the molecular mechanism underlying the pathogenesis of thymoma with or without MG. Methods: Molecular profiles of thymoma with or without MG were downloaded from The Cancer Genome Atlas, and Pearson's correlation analysis was performed to identify immune-related lncRNAs. T test was used to examine the differential expression and differential methylation between thymoma patients with or without MG. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to predict the function of target genes of immune-related lncRNAs. Results: Analyses of the 87 thymoma samples with complete MG information revealed that 205 mRNAs and 56 lncRNAs showed up-regulated expression in thymoma with MG patients, while 458 mRNAs and 84 lncRNAs showed down-regulated expression. The methylation level of three immune-related lncRNAs (AP000787.1, AC004943.1, WT1-AS, FOXG1-AS1) was significantly decreased in thymoma tissues, and the methylation level of these immune-related lncRNAs (WT1-AS: Cor = 0.368, p < 0.001; FOXG1-AS1: Cor = 0.288, p < 0.01; AC004943.1: Cor = -0.236, p < 0.05) correlated with their expression. GO and KEGG pathway analysis revealed that targets of the immune-related lncRNA FOXG1-AS1 were enriched in small GTPase binding and herpes simplex virus 1 infection. Transcription coregulator activity and cell cycle were the most enriched pathways for targets of lncRNA AC004943.1. LncRNA WT1-AS targets were most enriched in actin binding and axon guidance. Conclusion: Our results revealed the immune-related molecular profiling of thymoma with MG and without MG and identified key pathways involved in the underlying molecular mechanism of thymoma-related MG. These findings provide insights for further research of potential markers for thymoma-related MG.
Project description:Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG frequently develops in thymoma patients, the etiologic factors for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-sequencing, we identify ectopic expression of neuromuscular molecules in MG-type thymoma. These molecules are found within a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we name neuromuscular mTECs (nmTECs). MG-thymoma also exhibits microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell-cell interaction analysis also predicts the interaction between nmTECs and T/B cells via CXCL12-CXCR4. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma is further confirmed immunohistochemically and by cellular composition estimation from the MG-thymoma transcriptome. Altogether, this study suggests that nmTECs have a significant function in MG pathogenesis via ectopic expression of neuromuscular molecules.
Project description:IntroductionLabor-market participation is potentially very difficult for patients with refractory myasthenia gravis (MG). In this study, employment status and work absences are compared between refractory and nonrefractory MG.MethodsAdults (aged 18-64?years, all diagnosed ?2?years previously) were included if enrolled in the Myasthenia Gravis Foundation of America Patient Registry during July 2013 to February 2018.ResultsSeventy-six patients (9.2%) had refractory and 749 (90.8%) had nonrefractory disease; demographic data did not differ between groups. Relative to the nonrefractory group, the refractory group patients were more than twice as likely to work fewer hours per week (odds ratio [95% confidence interval]: currently employed, 2.777 [1.640-4.704]; employed over previous 6?months, 2.643 [1.595-4.380]), but those employed were not more likely to be absent from work.DiscussionBecause absence from the labor market adversely affects quality of life and personal finances, these findings reaffirm the considerable disease burden associated with refractory MG.
Project description:BackgroundThe Quantitative Myasthenia Gravis Score and the Myasthenia Gravis Composite are two commonly used outcome measures in Myasthenia Gravis. So far, their measurement properties have not been compared, so we aimed to study their psychometric properties using the Rasch model.Methods251 patients with stable myasthenia gravis were assessed with both scales, and 211 patients returned for a second assessment. We studied fit to the Rasch model at the first visit, and compared item fit, thresholds, differential item functioning, local dependence, person separation index, and tests for unidimensionality. We also assessed test-retest reliability and estimated the Minimal Detectable Change.ResultsNeither scale fit the Rasch model (X2p < 0.05). The Myasthenia Gravis Composite had lower discrimination properties than the Quantitative Myasthenia Gravis Scale (Person Separation Index: 0.14 and 0.7). There was local dependence in both scales, as well as differential item functioning for ocular and generalized disease. Disordered thresholds were found in 6(60%) items of the Myasthenia Gravis Composite and in 4(31%) of the Quantitative Myasthenia Gravis Score. Both tools had adequate test-retest reliability (ICCs >0.8). The minimally detectable change was 4.9 points for the Myasthenia Gravis Composite and 4.3 points for the Quantitative Myasthenia Gravis Score.ConclusionsNeither scale fulfilled Rasch model expectations. The Quantitative Myasthenia Gravis Score has higher discrimination than the Myasthenia Gravis Composite. Both tools have items with disordered thresholds, differential item functioning and local dependency. There was evidence of multidimensionality in the QMGS. The minimal detectable change values are higher than previous studies on the minimal significant change. These findings might inform future modifications of these tools.